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First published online 16 May 2007
doi: 10.1242/dev.001230

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Repression of Wnt/ß-catenin signaling in the anterior endoderm is essential for liver and pancreas development

Valérie A. McLin^*,, Scott A. Rankin^* and Aaron M. Zorn

Cincinnati Children's Research Foundation and Department of Pediatrics, College of Medicine, University of Cincinnati, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.

View larger version (81K): [in this window] [in a new window]   Fig. 1. Repression of ß-catenin signaling in the endoderm is necessary and sufficient for liver and pancreas development. (A) 32-cell stage Xenopus embryos were injected with either a pCSKA-Wnt8 plasmid (250 pg) or stabilized pt-ß-catenin RNA (250 pg) in the D1 anterior endoderm cells. Other embryos were injected with RNA encoding Dkk1 (500 pg) or Gsk3ß (500 pg) into D4 posterior endoderm cells to repress Wnt signaling. (B) In situ hybridization at stage 35 with the liver marker for1, or with a combination of pancreas/duodenum marker pdx1/xlhbox8 and the lung marker nkx2.1, or with the intestinal marker endocut. Some embryos were hybridized with just pdx1. Arrowheads indicate ectopic or repressed gene expression. The solid red line indicates the relative size of the foregut domain. Gut tubes were isolated at stage 42 to visualize organ bud morphology. The dashed red line outlines the liver bud. L, liver; P, pancreas; Lu, lungs. (C) In situ hybridization to Gsk3ß-injected guts with liver markers for1, ambp, the early pancreas marker ptf1a and the exocrine pancreas marker elastase. (D) A sectioned embryo co-injected with Gsk3ß and ß-gal RNA shows ß-gal-staining nuclei (blue) and for1 expression (brown) localized to the endoderm.

View larger version (103K): [in this window] [in a new window]   Fig. 2. Temporal regulation of ß-catenin/Tcf activity during endoderm pattering. (A) At the 32-cell stage, Xenopus embryos were injected in the anterior D1 cells with RNA encoding the fusion protein GR-LEFN-ßCTA (800 pg), which constitutively activates ß-catenin target genes in the presence of dexamethasone (Dex). Dex (1 µM) was added to the media of injected embryos at the indicated stages and embryos were assayed by for1, pdx1 and endocut in situ hybridization at stage 35. (B) Addition of Dex to GR-LEFN-ßCTA-injected embryos from stage 30 to 42, followed by hhex in situ, revealed enlarged liver buds. (C) 32-cell stage embryos were injected in posterior D4 cells with RNA encoding GR-NTcf3 (800 pg), which represses ß-catenin/Tcf target genes when activated. Dex (1 µM) was added to the media of injected embryos at the indicated stages and embryos were assayed by for1, pdx1 and endocut in situ hybridization at stage 35. (D) GR-NTcf3 was injected into D1 cells at the 32-cell stage, and when Dex was added from stages 30 to 42 some embryos exhibited smaller liver buds based on for1 in situ hybridization. No effect was observed in uninjected embryos treated with Dex.

View larger version (37K): [in this window] [in a new window]   Fig. 3. The endoderm is a direct target of ß-catenin signaling. (A) Experimental design of the endoderm transplantations. Anterior endoderm (AE) or posterior endoderm (PE) was dissected from gastrula Xenopus embryos injected with RNA encoding GFP, pt-ß-catenin+GFP or Gsk3ß and GFP and the tissue was transplanted into the bastocoel of uninjected sibling host embryos. (B,C) Confocal analysis at the neurula stage indicates that the GFP-labeled cells were incorporated into the host endoderm near the presumptive midgut. (D-G) At stage 42, control AE transplants (D) contributed primarily to liver (li) and foregut, whereas control PE transplants (E) mostly contributed to the intestine (i). By contrast, AE injected with pt-ß-catenin (F) rarely contributed to foregut, whereas PE injected with Gsk3ß (G) frequently contributed to the liver. GFP-labeled cells were only observed in the endoderm and not the heart (h) or other mesoderm tissue. (H) Bar chart showing the location frequency of each type of transplant.

View larger version (17K): [in this window] [in a new window]   Fig. 4. The cardiogenic mesoderm is required for liver specification. (A) At the 32-cell stage, Xenopus embryos were injected with Gsk3ß RNA (500 pg) into the D4 posterior endoderm cell. At stage 18, endoderm explants were isolated from control and injected embryos, and cultured with or without their associated mesoderm (orange) until stage 35 when they were assayed by RT-PCR. Foregut explants from uninjected embryos were either left intact with the anterior endoderm and associated mesoderm (AEM), or the anterior endoderm (AE, green) was separated from the mesoderm (AM). Posterior endoderm and mesoderm (PEM), or posterior endoderm without its associated mesoderm (PE), was also isolated from control and Gsk3ß-injected embryos (PE+Gsk3ß). (B,C) Bar charts showing the normalized relative mRNA expression levels from RT-PCR of the liver marker for1, heart marker cardiac-troponin and the pan-endodermal marker endodermin. WE, whole embryo; AE+AM, endoderm and mesoderm separated and immediately recombined.

View larger version (75K): [in this window] [in a new window]   Fig. 5. Regulation and function of Xenopus hhex. (A) Analysis of hhex expression by in situ hybridization to bisected stage-18 embryos (anterior left). (a) Schematic of a stage-18 bisected embryo showing the presumptive foregut (fg, green) and hindgut domain (hg). (b) Injection of GR-LEFN-ßCTA RNA (800 pg) into the D1 anterior endoderm cell has no effect without Dex. (c) Addition of Dex (1 µM) at the midgastrula repressed hhex expression as does (d) D1 injection of stabilized pt-ß-catenin RNA (250 pg). (e) Uninjected control embryo. (f) Injection of NTcf3 RNA (800 pg) or (g) Gsk3ß RNA (500 pg) in posterior D4 cells results in ectopic hhex expression (arrowhead). (h) Co-injection of Gsk3ß and ß-gal RNA reveals that the blue ß-gal stain co-localizes with ectopic hhex in the endoderm. (B) Hhex is required for liver and pancreas development. 32-cell stage embryos were injected with either an antisense hhex morpholino oligo (HexMO, 80 ng) in the D1 cells or with Gsk3ß or Gsk3ß plus HexMO in D4 cells. At stage 35, embryos were assayed by in situ hybridization with liver (for1) or pancreas/duodenum (pdx1) probes.

View larger version (60K): [in this window] [in a new window]   Fig. 6. Analysis of the Xenopus hhex promoter. (A,B) Confocal analysis of bisected hhex:gfp transgenic embryos at (A) midgastrula and (B) early somite stages (anterior left), showing GFP (green) in the anterior endoderm. Embryos were counterstained with anti-ß-catenin antibodies (red). (C-E) 50% of the foregut explants (AEM) isolated from embryos with a heterozygous hhex:gfp transgenic father were GFP-positive as expected (C), whereas posterior explants (PEM) did not express GFP (D). By contrast, 4 of 12 posterior explants isolated from sibling embryos injected with Gsk3ß RNA (500 pg) were GFP-positive (E). Upper panels, bright field images; bottom panels, GFP. (F) The indicated hhex:luciferase constructs were injected into D1 or D4 cells at the 32-cell stage and luciferase activity was assayed at the gastrula stage. (G) Bar chart showing results of F as normalized relative luciferase activity.

View larger version (98K): [in this window] [in a new window]   Fig. 7. Vent2 mediates ß-catenin function. (A) Xenopus embryos were injected with the indicated hhex:luciferase constructs with or without Vent2 RNA (500 pg) in D1 anterior or D4 posterior cells at the 32-cell stage. The bar chart shows the normalized relative luciferase activity at gastrula stage, indicating that Vent2 represses the hhex promoter. (B-D) In situ hybridization of bisected stage-18 embryos with the probes indicated. (E) Injection of Gsk3ß RNA (500 pg) in the posterior endoderm repressed vent2 expression. (F) Embryos were injected at the 32-cell stage with Vent2 RNA in anterior D1 cells or in posterior D4 cells with either Gsk3ß or Gsk3ß plus Vent2, followed by in situ hybridization at stage 18 with hhex, and stage 35 with for1 or pdx1 probes. (G) These data suggest a molecular pathway in which Wnt/ß-catenin signaling promotes vent2 expression and Vent2 represses hhex transcription.

View larger version (20K): [in this window] [in a new window]   Fig. 8. A model of Wnt/ß-catenin-mediated endoderm patterning in Xenopus. Schematic showing an early-somite stage Xenopus embryo. Canonical Wnts secreted from the mesoderm (orange) signal to the adjacent posterior endoderm (yellow) to repress foregut development by activating the homeodomain repressor Vent2, which in turn inhibits the expression of key foregut genes such as hhex and foxa2 in the posterior endoderm. The anterior endoderm (green) secretes a number of Wnt-antagonists that block the Wnt signals from the mesoderm, allowing hhex and foxa2 expression to impart foregut identity. The hhex-expressing anterior endoderm then sends an unknown signal to the adjacent mesoderm inducing it to become cardiac. Later in development, the cardiogenic mesoderm signals back to the endoderm (curved dashed line), inducing a subset of the foregut endoderm to adopt a hepatic fate.